For B.Pharm 5th semester

1. Tracer techniques,
Radioisotopes
used in
the investigation of Biogenetic
studies

2. Introduction
• Living plants considered as biosynthetic laboratory primary as well as
secondary metabolites.
• Different biosynthetic pathway:-
-Shikmic acid pathway
-Mevalonic acid pathway
-Amino acids pathway
• Various intermediate and steps are involved in biosynthetic pathway in
plants can be investigated by means of following techniques: -
• Tracer technique
• Use of isolated organ
• Grafting methods
• Use of mutant strain
 In this technique different isotope, mainly the radioactive
isotopes which are incorporated into presumed precursor of plant
metabolites and are used as marker in biogenic experiments.

3. Radioisotopes
• Radioisotopes are radioactive isotopes of an element.
• They can also be defined as atoms that contain an unstable combination of
neutrons and protons, or excess energy in their nucleus.
• The term isotope is formed from the Greek roots isos ("equal") and topos
("place"), meaning "the same place“.
• Isotope, one of two or more species of atoms of a chemical element with
the same atomic number and position in the periodic table and nearly
identical chemical behaviour but with different atomic masses and physical
properties.
• All isotopes of a given element have the same number of protons but
different numbers of neutrons in each atom.
• Every chemical element has one or more isotopes.
• It was coined by a Scottish doctor and writer Margaret Todd in 1913
Examples: Carbon-12, Carbon-13, and Carbon-14 are three isotopes of the
element Carbon with mass numbers 12, 13, and 14, respectively. The atomic
number of carbon is 6, which means that every carbon atom has 6 protons,
so that the neutron numbers of these isotopes are 6, 7, and 8 respectively.

4. More examples

5. Radioactive Decay
• An unstable atomic nucleus spontaneously loses
energy by emitting ionizing particles and
radiation.
Radioactive isotope Parent Nuclide Daughter
Nuclide
• When an unstable nucleus decays, it may give:
1) Alpha or helium Radiation.
2) Beta or Electron Radiation.
3) Gamma Radiation.

6. 1) Alpha particle decay
• Alpha particles are made up of 2 protons and 2
neutrons.
• Are same as helium nucleus.
• When a nucleus emits alpha particle, its atomic
number decreases by 2 and its atomic mass
decreases by 4.
• These particles are relatively slow and heavy.
• Low penetrating power.
• As they have large charge, alpha particles ionize
other atoms strongly.
• Alpha decay occurs in very heavy elements like
Uranium and Radium.

7. 2) Beta Particle Decay
• These particles are as same as electrons as they have
charge of minus 1.
• When a nucleus emits β particle the Atomic number
increases by 1 and Atomic mass is unchanged.
• They are fast and light.
• Have Medium Penetrating Power.
• Example of radioisotope emitting β, phosphorus-32.
• These particles ionize atoms that they pass, but not as
strongly as alpha particles do.

8. 3) Gamma rays
• Gamma rays are Waves not Particles.
• They have no mass and no charge.
• Atomic mass and number unchanged.
• High Penetrating Power.
• Do not directly ionize other atoms.
• We don’t find pure gamma source, they are
emitted along alpha or beta particles.
• Useful gamma source technetium-99, used as
tracer in medicine.

9. Penetration power of the Radiations

10. Half-life
• The half life of radioisotope is the time for the radiation level to decrease (decay) to
one half of the original value.
• Naturally occurring tend to have longer half lives.
• Used in nuclear medicine have short half lives.

11. Radiation Units
1. Curie (Ci)-measures activity as the number of atoms that
decay in one second.
1 Curie (Ci) = 3.7 × 1010 decays/sec.
2. rad (radiations absorbed dose)- Measures the radiation
absorbed by the tissues of the body.
1 rad = 0.01 Gy = 0.01 J/kg
3. rem (radiation equivalent mass)- Measures the biological
damage caused by different types of radiation.
1 rem = 0.01 Sievert
4. Becquerel (Bq) - 1Bq = 1 Disintegration per sec (dps)
5. Sievert (Sv) = 100 rem
6. Gray (Gy) = 1 J/Kg Tissue

12. Tracer technique
It can be defined as techniques which utilizes a
labelled compound to find or to trace the
different intermediates and various steps in
biosynthetic pathways in plants, at a given rate
and time.
OR
In this technique different isotope, mainly the
radioactive isotopes which are incorporated into
presumed precursor of plant metabolites and are
used as marker in biogenic experiments.

13. • The labelled compound can be prepared by use of two types of
isotopes.
i) Radioactive isotopes.
ii) Stable isotopes.
i) Radioactive isotopes: - [e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I
decay with emission of radiation] For biological investigation –
carbon & hydrogen.
• For metabolic studies – S, P, and alkali and alkaline earth
metals are used.
• For studies on protein, alkaloids, and amino acid–labelled
nitrogen atom give more specific information.
• 3H compound is commercially available.
ii) Stable isotopes: - [e.g. 2H, 13C, 15N, 18O] Used for labelling
compounds as possible intermediates in biosynthetic pathways.
• Usual method of detection are: – MASS spectroscopy [15N, 18O]
• NMR spectroscopy [2H, 13C]

14. Criteria for tracer techniques
• The starting concentration of tracer must be
sufficient withstand resistance with dilution in
course of metabolism.
• Proper Labelling:- for proper labelling physical
& chemical nature of compound must be
known.
• Labelled compound should involve in the
synthesis reaction.
• Labelled should not damage the system to
which it is used.

15. Requirement for tracer technique
1. Preparation of labelled compound.
2. Introduction of labelled compound
into a biological system.
3. Separation & determination of
labelled compound in various
biochemical fractions at later time.

16. 1.Preparation of Labelled Compound:-
 The labelled compound produce by growing chlorella in
atmosphere of 14CO2 All carbon compounds
14C labelled.
 The 3H (tritium) labelled compound are commercially
available. Tritium labelling is effected by catalytic
exchange in aqueous media by hydrogenation of
unsaturated compound with tritium gas. Tritium is pure β
emitter of low intensity & its radiation energy is lower
than 14C.
• By the use of organic synthesis:-

17. 2. Introduction of labelled compound:-
PRECAUTION:-
• The precursor should react at necessary site of synthesis in
plant.
• Plant at the experiment time should synthesize the compound
under investigation
• The dose given is for short period.
 Root feeding: From roots, roots are the main site for
biosynthesis.(Rauwolfia-alkaloids, )
 Stem feeding: Feeding is done from Stem, mainly for latex
containing plants(Rubber, indian hemp, etc)
 Direct injection: Administrate through injection, for hollow stem
plants(Umbeliferious, opium, etc)
 Infiltration: Suitable for rooted plant.
 Floating method: Achieved when there is small amount of material
is available
 Spray technique: by spraying method, steroid containing plants

18. 3. Separation and detection of compound:-
• Geiger–Muller counter.
• Liquid Scintillation counter.
• Gas ionization chamber.
• Bernstein–Bellentine counter.
• Mass spectroscopy.
• NMR eletrodemeter.
• Autoradiography.
• Radio paper chromatography.

19. Methods
1. PRECURSOR PRODUCT SEQUENCE:
• In this technique, the presumed precursor of the constituent under
investigation on a labelled form is fed into the plant and after a suitable
time the constituent is isolated, purified and radioactivity is determined.
Disadvantage:
• The radioactivity of isolated compound alone is not usually sufficient
evidence that the particular compound fed is direct precursor, because
substance may enter the general metabolic pathway and from there may
become randomly distributed through a whole range of product.
Application:
• Stopping of hordenine production in barley seedling after 15–20 days
of germination.
• Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura
stramonium.
• This method is applied to the biogenesis of morphine & ergot alkaloids.

20. 2. DOUBLE & MULTIPLE LABELLING:-
• This method give the evidence for nature of biochemical
incorporation of precursor arises double & triple labelling.
• In this method specifically labelled precursor and their subsequent
degradation of recover product are more employed.
• Application: -
• This method is extensively applied to study the biogenesis of plant
secondary metabolite.
• Used for study of morphine alkaloid. E.g. Leete, use Doubly labelled
lysine used to determine which hydrogen of lysine molecule was
involved in formation of piperidine ring of anabasine in Nicotina
glauca.

21. 3. COMPETITIVE FEEDING:
• If incorporation is obtained it is necessary to consider whether this infact, the
normal route of synthesis in plant not the subsidiary pathway.
• Competitive feeding can distinguish whether B & B’ is normal intermediate
in the formation of C from A.
Application: -
• This method is used for elucidation of biogenesis of propane alkaloids.
• Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis
of alkaloids of Conium maculactum (hemlock) using 14C labelled
compounds.

22. 4. ISOTOPE INCORPORATION:
• This method provides information about the position of
bond cleavage & their formation during reaction.
• E.g. Glucose – 1- phosphatase cleavage as catalyzed by
alkaline phosphatase this reaction occur with cleavage of
either C – O bond or P – O bond.

23. 5. SEQUENTIALANALYSIS:
• The principle of this method of investigation is to grow
plant in atmosphere of 14CO2 & then analyze the plant at
given time interval to obtain the sequence in which various
correlated compound become labelled.
Application:-
• 14CO2 & sequential analysis has been very successfully
used in elucidation of carbon in photosynthesis.
• Determination of sequential formation of opium hemlock
and tobacco alkaloids.
• Exposure as less as 5 min. 14CO2, is used in detecting
biosynthetic sequence as-
• Piperitone --------- (-) Menthone ---------- (-) Menthol in
Mentha piperita.

24. Application of tracer technique
 Study of squalene cyclization by use of 14C, 3H labelled mevalonic
acid.
 Interrelationship among 4 – methyl sterols & 4, 4 dimethyl sterols, by
use of 14C acetate.
 Terpenoid biosynthesis by chloroplast isolated in organic solvent, by
use of 2- 14C mevalonate.
 Study the formation of cinnamic acid in pathway of coumarin from
labelled coumarin.
 Origin of carbon & nitrogen atoms of purine ring system by use of
14C or 15N labelled precursor.
 Study of formation of scopoletin by use of labelled phenylalanine.
 By use of 45Ca as tracer, - found that the uptake of calcium by plants
from the soil. (CaO & CaCO2).
 By adding ammonium phosphate labelled with 32P of known specific
activity the uptake of phosphorus is followed by measuring the
radioactivity as label reaches first in lower part of plant, than the
upper part i.e. branches, leaves etc.

25. Advantages
High sensitivity.
Applicable to all living organism.
Wide ranges of isotopes are available.
More reliable, easily administration &
isolation procedure.
Gives accurate result, if proper metabolic time
& technique applied.

26. Disadvantages
Kinetic effect
Chemical effect
Radiation effect
Radiochemical purity
High concentration distorting the result.